In many techniques of molecular biology, it is important to have samples of high quality. Results are generally enhanced in PCR, sequencing, fragment analysis, and so forth, when the subject bio-molecule materials are separated from potentially interfering contaminants. Thus, it is often desirable to purify/concentrate the bio-molecules (e.g., polynucleotides, such as DNA, RNA, PNA, etc.) of interest in samples prior to analysis.
In analyses utilizing laser-induced fluorescence (LIF) detection techniques, typical DNA samples often contain, in addition to dye-labeled DNA: salts, residual enzyme, DNA oligonucleotides, dNTP's, dye-labeled ddNTP's, and/or surfactants. It is generally desirable to remove all species except the subject dye-labeled DNA fragments. However, even partial purification can be useful. Thus, at a minimum, it is often desirable to remove at least some of the species that are present at higher concentration and that could interfere with the analysis.
Sample concentration can be used to improve the detection limits of various analytical methods, such as electrophoresis. For example, the starting zone length of a sample injection can be reduced by utilization of a process termed “stacking.” Stacking reduces the width of the sample zone before separation, which can result in improved sensitivity and increased peak efficiency.
Xiong et al. describe a method for pH-mediated sample concentration of DNA sequencing samples on a capillary tube. While the technique of Xiong et al. might allow for sufficient signal from direct load on unpurified sequencing samples, it would not be expected to remove unincorporated dyes and contaminants that can obscure the sequencing data. Briefly, according to the method of Xiong et al., a capillary is filled with a nucleic acid DNA separation polymer. However, the polymer solution is buffered with a basic buffer that is charged at low pH but neutralized at high pH. Xiong et al. employed Tris buffer for this purpose. The first stage of the process involves a very long electrokinetic injection from unpurified sequencing reaction (e.g., right off a thermocycler with no following cleanup step). Because the sample is very salty at this point, the electrokinetic injection process is inefficient and a long injection time is needed to move enough DNA into the capillary to obtain sufficient signal. The injection time is so long that the peaks would be far too broad to achieve the necessary resolution for DNA sequencing. To re-focus the DNA starting band, Xiong et al. follow the DNA injection with a period of electrophoresis from a sodium hydroxide solution. The hydroxide migrates into the capillary, neutralizing the Tris buffer as it enters. In the area where the Tris is neutralized the conductivity becomes very low and therefore the electric field increases. The increased electric field at the injection end of the capillary allows the DNA at the back of the injection plug to travel faster than the DNA at the front of the injection plug. This refocuses the injection plug and allows for reasonable resolution to be obtained. When this technique is used with standard capillary electrophoresis, the contaminating dye labeled terminators, which are present in much higher concentration than the DNA, also migrate into the separation capillary. The large concentration of dye can migrate with the DNA and may negatively impact some section of the sequencing data.